Lentiviral Production Using X-tremeGENE HP Transfection Reagent

Lentiviruses represent a powerful tool in research applications to transduce a wide range of cell types. During virus production, low titers and toxicity can be challenging. This easy-to-use protocol uses X-tremeGENE HP Transfection Reagent for fast and robust lentiviral preparation, with high virus titers being suitable to successfully transduct target cells.

Materials

Packaging Cell Line
TLA-HEK293T (Open Biosystems) This cell line grows adherently and is amenable to serumfree media formulations. When using X-tremeGene HP reagent, adaption to serum-free conditions is not required since this reagent is not impaired by the presence of serum. The cells should be cultured in Opti-MEMR with GlutaMAX (Invitrogen) and 10% fetal bovine serum (FBS). Maintain culture vessels at 37°C at 10% CO2 in 95% RH.

Results

To determine whether this protocol produced sufficiently pure and concentrated virus suitable for cell culture applications, cells were transfected according to the procedure described above. Microscopy analysis clearly revealed high levels of GFP expression (<90%) without toxicity (Figure 1).

Furthermore, the microscopy analyses following transduction using the produced virus-containing supernatant revealed yields of GFP-positive cells at 1.2 x 106 pfu/ml. Thus, the protocol established here allows the production of lentiviral viruses in suitable amounts. To avoid multiple insertions of virus DNA into the host genome, it is recommended to infect the cells at a multiplicity of infection (MOI) below 0.1. This protocol provides virus amounts derived from one well of a standard 6-well plate for the transduction of at least 107 cells.

For life science research only. Not for use in diagnostic procedures.

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